Name: isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement
Uploaded: 2017-07-26T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

This work was supported by the Swiss National Science Foundation (grant number 310030-166313), The "Fondation Suisse pour la recherche sur les maladies musculaires," the "Foundation Marcel Levaillant," and the "Fondation pour la recherche ost&#233;o-articulaire."

Human muscle samples (tissues obtained from semitendinous muscles) were collected during orthopedic surgery on healthy patients as surgical waste. All methods relating to the human study were performed in accordance with the guidelines and regulations of the Swiss Regulatory Health Authorities and approved by the Commission Cantonale d&#39;Ethique de la Recherche from the Geneva Cantonal Authorities, Switzerland (protocol CER n&#176; 12-259). Informed and written consents were obtained from all adult subjects involved in...

<ol>
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The isolation and culture of human myoblasts from adult skeletal muscle offers an in vitro model to study muscle differentiation and muscle regeneration. In this paper, we provide a protocol that allows for the purification of high yields of human myoblasts in a simple and cost-limited way. In addition, this technique provides reliable and reproducible results in terms of percentage of myoblasts isolated and their myogenic differentiation efficiency. Indeed, we obtained around 60...

Human skeletal muscles are composed of groups of contractile, multinucleated muscle fibers resulting from the fusion of myogenic precursor cells. Skeletal muscles have the capacity to regenerate after injury thanks to the presence of SCs, the skeletal muscle stem cells located between the plasma membrane of myofibers (sarcolemma) and the basal lamina. In uninjured muscle, SCs are mostly present in a quiescent state. In response to mechanical stress or injury, SCs become activated (myoblasts), proliferate, and undergo either differentiation to form new myofibers or self-renewal to replenish the SC pool1,2. Over the years, several techniques were developed to isolate SCs and their progeny, the myoblasts, from skeletal muscle biopsies. With the greater understanding of the cell surface markers expressed on these cells and the methodology of fluorescence-activated cell sorting (FACS)3,4,5, it is now possible to isolate pure populations of human myoblasts from muscle samples.
Calcium signaling regulates skeletal muscle development, homeostasis, and regeneration. In particular, the Ca2+ entry that is activated following intracellular store depletion, called SOCE, is of great importance6 for these processes. Upon cell stimulation, the Ca2+ level in the endo/sarcoplasmic reticulum (ER/SR) decreases, which in turn activates plasma membrane Ca2+ channels that permit Ca2+ entry to refill the ER/SR7. The two main proteins involved in SOCE are the ER/SR Ca2+-sensing stromal interaction molecule 1 (STIM1) protein and the plasma membrane Ca2+ channel Orai1. Skeletal muscle abundantly expresses these two proteins, as well as other proteins of the same families (STIM2 and Orai2-Orai3)8,9 and several Ca2+-permeable channels of the transient receptor potential canonical (TRPC) family10,11,12,13. Ca2+ entry through the SOCE pathway is of great importance for muscle formation/regeneration14. Mutations of STIM1 or Orai1 proteins have deleterious effects on muscle function, leading mainly to muscle hypotonia15. Animal models with SOCE impairment also display reduced muscle mass and force, together with enhanced fatigability6,16,17. As mentioned, other STIM and Orai proteins, as well as many TRPC channels, are expressed in skeletal muscle, and their respective roles have not been determined so far. By knocking down their expression level, it is thus possible to investigate their implications in SOCE and their roles during human skeletal muscle differentiation.
SOCE measurement can be performed using two different approaches: electrophysiological current recordings and Ca2+ measurements. Electrophysiology is certainly a more direct method, as it measures the current of interest and its associated electrophysiological signature. However, this technique is very difficult to apply to muscle cells, mainly because of the large size of the muscle cells and the small size of the endogenous SOCE current. Cytosolic Ca2+ imaging is technically very accessible, but the measure is more indirect, as the Ca2+ level measured in the cytosol reflects both the entry of Ca2+ and the re-pumping out of the cell or in the internal stores.
A methodology that includes isolating myoblasts from small pieces of human skeletal muscle after enzymatic digestion, amplification, and FACS is explained in this paper. The process of muscle differentiation and the immunofluorescence protocol to follow the expression of differentiation markers over time are described18. Finally, the measurement of SOCE and the role of different ion channels in Ca2+ signaling and skeletal muscle differentiation are explained.

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			<td height="25" style="height:25px;width:321px;">Ham&#8217;s F10</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">31550-023</td>
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			<td height="25" style="height:25px;width:321px;">Gentamycin</td>
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			<td height="25" style="height:25px;width:321px;">Trypsin-EDTA 0.05%</td>
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			<td height="25" style="height:25px;width:321px;">70 &#956;m cell strainer</td>
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			<td style="width:235px;">Falcon</td>
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			<td height="25" style="height:25px;width:321px;">Falcon 5&#160;ml tube (FACS)</td>
			<td style="width:235px;">Falcon</td>
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			<td height="25" style="height:25px;width:321px;">Culture medium: OPTI-MEM</td>
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			<td height="25" style="height:25px;width:321px;">Mouse anti-MyHC *</td>
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			<td style="width:201px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">DAPI</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">D9542</td>
			<td style="width:201px;">CAUTION:&#160; toxic compound</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Paraformaldehyde</td>
			<td style="width:235px;">Fluka</td>
			<td style="width:187px;">762400</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Fura-2 AM</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">F1201</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Pluronic F-127</td>
			<td style="width:235px;">ThermoFisher Scientific</td>
			<td style="width:187px;">P3000MP</td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">Thapsigargin</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:187px;">T9033</td>
			<td style="width:201px;">CAUTION&#160;: toxic compound&#160;</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:321px;">Ratio imaging acquisition software: Metafluor 6.3</td>
			<td style="width:235px;">Molecular Device</td>
			<td style="width:187px;"></td>
			<td style="width:201px;"></td>
		</tr>
		<tr height="24">
			<td colspan="2" height="24" style="height:24px;">*&#160;: MF 20 was deposited to the DSHB by Fischman, D.A.&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of human myoblasts, assessment of myogenic differentiation, and store-operated calcium entry measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

After the enzymatic dissociation of a human muscle sample, cells were amplified in GM. Human myoblasts, defined as CD56+/CD146+/CD45-/CD34-/CD144- cells, were obtained after FACS. Myoblasts represented more than 60% of the analyzed population (Figure 1). Primary human myoblasts were replated, grown to confluence, and cultured in DM for 48 h. After immunostaining for the expression of the transcription factor MEF2 and the muscle-specific protein myosin heavy chain (MyHC), we observed that a m...

Watch this Scientific Journal Video about Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement at JoVE.com

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

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